Localization of the retinal protonated Schiff base counterion in rhodopsin

Abstract

Semiempirical molecular orbital calculations are combined with 13C NMR chemical shifts to localize the counterion in the retinal binding site of vertebrate rhodopsin. Charge densities along the polyene chain are calculated for an 11-cis-retinylidene protonated Schiff base (11-cis-RPSB) chromophore with 1) a chloride counterion at various distances from the Schiff base nitrogen, 2) one or two chloride counterions at different positions along the retinal chain from C10 to C15 and at the Schiff base nitrogen, and 3) a carboxylate counterion out of the retinal plane near C12. Increasing the distance of the negative counterion from the Schiff base results in an enhancement of alternating negative and positive partial charge on the even- and odd-numbered carbons, respectively, when compared to the 11-cis-RPSB chloride model compound. In contrast, the observed 13C NMR data of rhodopsin exhibit downfield chemical shifts from C8 to C13 relative to the 11-cis-RPSB.Cl corresponding to a net increase of partial positive or decrease of partial negative charge at these positions (Smith, S. O., I. Palings, M. E. Miley, J. Courtin, H. de Groot, J. Lugtenburg, R. A. Mathies, and R. G. Griffin. 1990. Biochemistry. 29:8158-8164). The anomalous changes in charge density reflected in the rhodopsin NMR chemical shifts can be qualitatively modeled by placing a single negative charge above C12. The calculated fit improves when a carboxylate counterion is used to model the retinal binding site. Inclusion of water in the model does not alter the fit to the NMR data, although it is consistent with observations based on other methods. These data constrain the location and the orientation of the Glu113 side chain, which is known to be the counterion in rhodopsin, and argue for a strong interaction centered at C12 of the retinylidene chain.

Functional Interaction of Transmembrane Helices 3 and 6 in Rhodopsin

“…The results show that specific portions of TM helices 3 and 6 in rhodopsin comprise specific retinal-protein contacts that define the chromophore-binding pocket. The present results are consistent with a model of rhodopsin based on data from electron microscopy studies (6), NMR and two-photon spectroscopy measurements (7)(8)(9), and a comparative analysis of G protein-coupled receptors (10). A molecular graphics model of the transmembrane domain of rhodopsin is presented that illustrates the relative proximity of the retinal chromophore to Gly 121 and Phe 261 .…”

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

“…We rotated 11-cis-retinal along the retinal axis to a position where the main band, looking from the Schiff base (SB) end to the ␤-ionone ring, points 16°out of the membrane plane toward the intradiscal side and the cis band points 22°out of the membrane plane in the opposite direction (toward the cytoplasmic side). This results in a rotation angle of Ϸ40-50°of the retinal plane (defined as C 6 to C 12 ) relative to the membrane plane and places Glu-113 below the retinal axis in a position proposed previously (6,7). This initial position of 11-cis-retinal in the dark state reduces the 16 different possible configurations determined in ref.…”

“…22 to four possible configurations. The distance of C 12 to O 1 of Glu-113 was taken to be Ϸ3 Å (6,7,41).…”

“…Absorption of light transforms rhodopsin (rho) into an activated state, which interacts with a heterotrimeric G protein to initiate an enzyme cascade leading to visual transduction. To reach this active state, called metarhodopsin (meta) II (1,2), rho passes through a number of intermediates that have been characterized by UV-visible, Fourier-transform infrared (FTIR), Raman, and NMR spectroscopies, in addition to spin-labeling and biochemical studies (3)(4)(5)(6)(7)(8)(9)(10). One way to characterize the intermediates is to trap them at low temperatures.…”

Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: Expression of 15 N-lysine- and 13 C-glycine-labeled opsin in a stable cell line

“…As a result, the ''salt bridge'' in rhodopsin is clearly not a simple ion pair. Chemical shift measurements of the protein-bound retinal chromophore previously were used to localize the Glu-113 interaction near C12 of the retinylidene chain (17,34). Yet, this position of the counterion appears to be inconsistent with the high C ϭ N stretching frequency of the retinal Schiff base linkage that is similar to 11-cis retinal PSB model compounds in methanol and has long been interpreted in terms of a strong Schiff base hydrogen bond (35).…”